1. Field of the Invention
The present invention relates to peptide antagonists which specifically prevent laminin interaction with nidogen.
2. Background of the Invention
Various isoforms of cross-shaped laminins have been identified as major cell-adhesive and structural proteins of basement membranes and other extracellular structures (Engel, J. (1993), In Molecular and Cellular Aspects of Basement Membranes, Rohrbach, D. H. and Timpl, R., (eds), Academic Press, San Diego, Calif., pp. 147-176; Timpl, R. and Brown, J. C. (1994), Matrix Biol.). They are large multidomain proteins (600-900 kDa) and consist of disulfide-linked .alpha., .beta. and .gamma. chains (for recent nomenclature see Burgeson, R. E., et al. (1994) Matrix Biol, 14, 209-211; Timpl, R. and Brown, J. C. (1994), Matrix Biol.). Many heterotypic interaction sites have been demonstrated for laminin 1 of chain composition .alpha.1.beta.1.gamma.1, including a single high affinity binding side (.kappa..sub.D =0.5 nM) for the 150 kDa basement membrane protein nidogen (Fox, J. W. et al. (1991), EMBO J., 10, 3137-3146). Nidogen also binds to collagen IV, the proteoglycan perlecan and other extracellular ligands and thus mediates the formation of ternary complexes between laminin 1 and other components (Mayer, U. and Timpl, R. (1994) In Extracellular Matrix Assembly and Structure, Yurchenco, P. D., Birk, D. and Mecham, R. P. (eds), Academic Press, Orlando, Fla. pp. 389-416; Brown, J. C. et al. (1994), J. Cell Sci., 107, 329-338). Nidogen binding to laminins seems therefore to be a critical step in the supramolecular assembly of basement membranes. This interpretation was recently underscored in studies with antibodies which block the nidogen binding site of laminin 1 (Mayer, U. et al. (1993), EMBO J., 12, 1879-1885) and inhibit kidney tubulogenesis and lung branching in embryonic organ cultures (Ekblom, P. et al (1994) Development, 120, 2003-2014).
The high affinity nidogen binding site has been localized to a single motif homologous to epidermal growth factor (EGF) present in the short arm domain III of the mouse laminin .gamma.1 chain (Gerl, M. et al. (1991), Eur. J. Biochem., 292, 167-174; Mayes, U. et al (1993) EMBO J., 12, 1879-1885). This laminin EGF-like repeat .gamma.1III4 consists of 56 residues and of four disulfide-linked loops (a-d), as indicated from the homology to EGF (Cooke, R. M. et al. (1987) Nature, 327, 339-341; Montelione, G. T. et al. (1987) Proc. Natl. Acad. Sci. USA, 84, 5226-5230) and other representative sequence features (Sasaki, M. and Yamada, Y. (1987), J. Biol. Chem., 262, 17111-17117; Engel, J. (1989), FEBS Lett., 251, 1-7). The same affinity for nidogen binding was also observed with human laminins 2 and 4, of chain compositions .alpha.2.beta.1.gamma.1 and .alpha.2.beta.1.gamma.1 (Brown, J. C. et al. (1994), J. Cell Sci., 107, 329-338), and is explained by the 97% sequence identity of the mouse and human .gamma.1III4 structure (Pikkarainen, T. et al. (1987), J. Biol. Chem., 263, 6751-6758). A lower sequence identity (61%) has been shown for this repeat in Drosophila laminin .gamma.1 chain (Chi, H. C. and Hui, C. F. (1989), J. Biol Chem., 264, 1543-1550) and the human laminin .gamma.2 chain isoform (77%; Kallunki, P. et al. (1992), J. Cell Biol., 119, 679-693). This has raised the question of whether these laminins also have affinity for nidogen and makes the more precise mapping of binding structures in the mouse repeat .gamma.1III4 necessary for molecular interpretations.
Despite the huge number of EGF-like repeats identified in many extracellular and membrane-bound proteins (see Roes, D. J. G. et al. (1988), EMBO J., 7, 2053-2061; Selander-Sunnerhagen, M. et al. (1992), J. Biol. Chem., 267, 19642-19649), little is known about their binding properties and the structures involved. Biological evidence for the importance of these repeats comes from studies of fibrilin mutants which are considered to cause Marfan syndrome (Dietz H. C. et al. (1991) Nature, 352, 337-339; Lee, B. et al. (1991) Nature, 352, 330-334) and from lethal mutations in the neurogenic Drosophila protein notch (Kelley, M. R. et al. (1987) Cell, 51, 539-548), but the functional basis is not yet understood. A precise identification has, however, been achieved for calcium binding sequences present in loop a and interloop regions of EGF-like repeats from several coagulation factors (Handford, P. A. et al. (1991), Nature, 351, 164-167; Selander-Sunnerhagen, M. et al. (1992), J. Biol. Chem., 267, 19642-19649). Site directed mutagenesis was also used to map the receptor binding site of EGF and demonstrated crucial residues at the C-terminal end beyond loop c and in the hinge region between loops b and c (Moy, F. J. et al. (1989), Proc. Natl. Acad. Sci, USA., 86, 9836-9840; Campion, S. R. et al. (1992) J. Cell Biochem, 50, 35-42; Campion, S. R. et al. (1993) Protein Engng., 6, 651-659).
In view of the potential importance of nidogen binding to laminin, it is desirable to provide peptide antagonists which inhibit laminin interaction with nidogen.